JB Accepts, published online ahead of print on 6 November 2009

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J. Bacteriol. doi:10.1128/JB.00935-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Biochemical and domain analyses of FSUAxe6B, a modular acetyl xylan esterase, yields a unique carbohydrate binding module from Fibrobacter succinogenes S85

Shosuke Yoshida, Roderick I. Mackie, and Isaac K.O. Cann^*

Energy Biosciences Institute, Institute for Genomic Biology, Department of Animal Sciences, and the Department of Microbiology, University of Illinois, Urbana, IL 61801, USA

^* To whom correspondence should be addressed. Email: icann{at}illinois.edu.

Acetylxylan esterase (E.C. 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of -1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetylxylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Homologous sequences that are similar to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module-1 (FPm-1). The FPm-1 modules are associated with at least 24 polypeptides in the genome of F. succinogenes S85. Bioinformatics search showed that most of the FPm-1 appended polypeptides are putative carbohydrate active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies on highly conserved active site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser₄₄, His₂₇₃, Glu₁₉₄ and Asp₂₇₀, with both Glu₁₉₄ and Asp₂₇₀ functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.